LOCUS       Exported                8180 bp ds-DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pACYC_synB_cstII_lgtB_ApNGT, complete sequence.
ACCESSION   KY212112
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8180)
  AUTHORS   Keys TG, Wetter M, Hang I, Rutschmann C, Russo S, Mally M, Steffen 
            M, Zuppiger M, Muller F, Schneider J, Faridmoayer A, Lin CW, Aebi M.
  TITLE     A biosynthetic route for polysialylating proteins in Escherichia 
            coli
  JOURNAL   Metab. Eng. (2017) In press
  PUBMED    29101090
REFERENCE   2  (bases 1 to 8180)
  AUTHORS   Keys TG.
  TITLE     Direct Submission
  JOURNAL   Submitted (21-NOV-2016) Institute of Microbiology, ETH Zurich, 
            Vladimir-Prelog-Weg 4, Zurich, ZH 8093, Switzerland
REFERENCE   3  (bases 1 to 8180)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..8180
                     /organism="Expression vector pACYC_synB_cstII_lgtB_ApNGT"
                     /mol_type="other DNA"
                     /label=PMA991
                     /note="PMA991"
                     /db_xref="taxon:2045053"
     regulatory      28..75
                     /regulatory_class="terminator"
                     /note="T7 terminator"
     terminator      28..75
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(295..954)
                     /codon_start=1
                     /product="CmR"
                     /label=CmR
                     /protein_id="ATN32178.1"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(955..1057)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     misc_feature    1316..2228
                     /label=ori - P15A
                     /note="ori - P15A"
     rep_origin      complement(1583..2127)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     CDS             complement(2352..3434)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     promoter        complement(3435..3512)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        3550..3580
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     regulatory      3550..3555
                     /regulatory_class="minus_35_signal"
     regulatory      3574..3580
                     /regulatory_class="minus_10_signal"
                     /note="-10 seq (TATAAT; Pribnow box)"
     protein_bind    3584..3608
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     misc_feature    3586..3606
                     /label=lac operator
                     /note="lac operator"
     CDS             3656..4342
                     /codon_start=1
                     /product="SynB"
                     /label=SynB
                     /protein_id="ATN32179.1"
                     /translation="MEKQNIAVILARQNSKGLPLKNLRKMNGISLLGHTINAAISSKCF
                     DRIIVSTDGGLIAEEAKNFGVEVVLRPAELASDTASSISGVIHALETIGSNSGTVTLLQ
                     PTSPLRTGAHIREAFSLFDEKIKGSVVSACPMEHHPLKTLLQINNGEYAPMRHLSDLEQ
                     PRQQLPQAFRPNGAIYINDTASLIANNCFFIAPTKLYIMSHQDSIDIDTELDLQQAENI
                     LNHKES"
     promoter        4357..4387
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     regulatory      4357..4362
                     /regulatory_class="minus_35_signal"
     regulatory      4381..4387
                     /regulatory_class="minus_10_signal"
                     /note="-10 seq (TATAAT; Pribnow box)"
     protein_bind    4391..4415
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     misc_feature    4393..4413
                     /label=lac operator
                     /note="lac operator"
     CDS             4463..5242
                     /codon_start=1
                     /product="CstII"
                     /label=CstII
                     /note="delta-C32 and I53S mutation"
                     /protein_id="ATN32180.1"
                     /translation="MKKVIIAGNGPSLKEIDYSRLPNDFDVFRCNQFYFEDKYYLGKKC
                     KAVFYNPSLFFEQYYTLKHLIQNQEYETELIMCSNYNQAHLENENFVKTFYDYFPDAHL
                     GYDFFKQLKDFNAYFKFHEIYFNQRITSGVYMCAVAIALGYKEIYLSGIDFYQNGSSYA
                     FDTKQKNLLKLAPNFKNDNSHYIGHSKNTDIKALEFLEKTYKIKLYCLCPNSLLANFIG
                     LAPNLNSNFIIQEKNNYTKDILIPSSEAYGKFSKNIN"
     promoter        5254..5284
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     regulatory      5254..5259
                     /regulatory_class="minus_35_signal"
     regulatory      5278..5284
                     /regulatory_class="minus_10_signal"
                     /note="-10 seq (TATAAT; Pribnow box)"
     protein_bind    5288..5312
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     misc_feature    5290..5310
                     /label=lac operator
                     /note="lac operator"
     CDS             5360..6190
                     /codon_start=1
                     /product="LgtB"
                     /label=LgtB
                     /protein_id="ATN32181.1"
                     /translation="MSQNHVISLASAAERRAHIADTFGRHGIPFQFFDALMPSERLEQA
                     MAELVPGLSAHPYLSGVEKACFMSHAVLWKQALDEGLPYITVFEDDVLLGEGAEKFLAE
                     DAWLQERFDPDTAFIVRLETMFMHVLTSPSGVADYCGRAFPLLESEHWGTAGYIISRKA
                     MRFFLDRFAALPPEGLHPVDLMMFSDFFDREGMPVCQLNPALCAQELHYAKFHDQNSAL
                     GSLIEHDRLLNRKQQRRDSPANTFKHRLIRALTKISREREKRRQRREQFIVPFQ"
     promoter        6202..6232
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     regulatory      6202..6207
                     /regulatory_class="minus_35_signal"
     regulatory      6226..6232
                     /regulatory_class="minus_10_signal"
                     /note="-10 seq (TATAAT; Pribnow box)"
     protein_bind    6236..6260
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     misc_feature    6238..6258
                     /label=lac operator
                     /note="lac operator"
     CDS             6308..8170
                     /codon_start=1
                     /product="ApNGT"
                     /label=ApNGT
                     /protein_id="ATN32182.1"
                     /translation="MENENKPNVANFEAAVAVKDYEKACSELLLILSQLDSNFGGIQEI
                     EFEYPVQLQDLEQEKIVYFCTRMATAITTLFSDPVLEISDLGVQRFLVYQRWLALIFAS
                     SPFVNADHILQTYNREPNRKNSLEIHLDSSKSSLIKFCILYLPESNVNLNLDVMWNISP
                     ELCASLCFALQSPRFIGTSTAFNKRATILQWFPRHLDQLKNLNNIPSAISHDVYMHCSY
                     DTSVNKHDVKRALNHVIRRHIESEYGWKDRYVAHIGYRNNKPVMVVLLEHFHSAHSIYR
                     THSTSMIAAREHFYLIGLGSPSVDQAGQEVFDEFHLVAGDNMKQKLEFIRSVCESNGAA
                     IFYMPSIGMDMTTIFASNTRLAPIQAIALGHPATTHSDFIEYVIVEDDYVGSEACFSET
                     LLRLPKDALPYVPSALAPEKVDYLLRENPEVVNIGIASTTMKLNPYFLEALKAIRDRAK
                     VKVHFHFALGQSNGITHPYVERFIKSYLGDSATAHPHSPYHQYLRILHNCDMMVNPFPF
                     GNTNGIIDMVTLGLVGVCKTGAEVHEHIDEGLFKRLGLPEWLIANTVDEYVERAVRLAE
                     NHQERLELRRYIIENNGLNTLFTGDPRPMGQVFLEKLNAFLKEN"